Measuring systems for biochemical analytics are important components of clinically relevant analytical methods. This primarily concerns the measurement of analytes e.g. metabolites or substrates which are determined directly or indirectly with the aid of an enzyme. The analytes are converted with the aid of an enzyme-coenzyme complex and subsequently quantified. In this process the analyte to be determined is contacted with a suitable enzyme and a coenzyme where the enzyme is usually used in catalytic amounts. The coenzyme is changed e.g. oxidized or reduced by the enzymatic reaction. This process can be detected electrochemically or photometrically either directly or by means of a mediator. A calibration yields a direct correlation between the measured value and the concentration of the analyte to be determined.
Coenzymes are organic molecules which are covalently or non-covalently bound to an enzyme and are changed by the conversion of the analyte. Prominent examples of coenzymes are nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) from which NADH or NADPH respectively are formed by reduction.
Measuring systems known from the prior art are characterized by a limited shelf-life and by special requirements for the environment such as cooling or dry storage in order to achieve this storage stability. Hence erroneous results caused by incorrect, unnoticed, faulty storage can occur for certain forms of application, e.g. in the cases of tests which are carried out by the end-users themselves such as blood glucose self-monitoring. Especially the exhaustion of desiccants due to opening the primary packaging for excessive periods can result in measuring errors which in some systems can be hardly recognized by the consumer.
A known measure that can be used to increase the stability of biochemical measuring systems is the use of stable enzymes, e.g. the use of enzymes from thermophilic organisms. It is also possible to stabilize enzymes by chemical modification, e.g. cross-linking or by mutagenesis. Furthermore, enzyme stabilizers such as trehalose, polyvinylpyrrolidone and serum albumin can also be added or the enzymes can be enclosed in polymer networks, e.g. by photopolymerization.
It has also been attempted to improve the storage life of biochemical measuring systems by using stable mediators. Thus the specificity of tests is increased and interferences during the reaction are eliminated by using mediators having the lowest possible redox potential. However, the redox potentials of the enzyme/coenzyme complexes constitutes a lower limit for the redox potential. If those fall below this limit, the reaction with the mediators is slowed down or even prevented.
Alternatively it is also possible to use biochemical measuring systems without mediators in which for example coenzymes such as the coenzyme NADH are directly detected. However, a disadvantage of such measuring systems is that coenzymes such as NAD and NADP are unstable.
NAD and NADP are base-labile molecules, the degradation paths of which are described in the literature (N. J. Oppenheimer in The Pyridine Nucleotide Coenzymes Academic Press, New York, London 1982, J. Everese, B. Anderson, K. You, Editors, chapter 3, pages 56-65). Essentially ADP-ribose is formed during the degradation of NAD or NADP by cleavage of the glycosyl bonds between the ribose and the pyridine unit. The reduced forms NADH and NADPH are, however, acid labile. For example, epimerization is a known degradation path. In both cases, the instability of NAD/NADP and NADH/NADPH is due to the liability of the glycosyl bonds between the ribose and the pyridine unit. But even under less drastic conditions such as in aqueous solution, the coenzymes NAD or NADP are already hydrolyzed solely by the ambient humidity. This instability can result in inaccuracies when measuring analytes.
A number of NAD/NADP derivatives are described for example in B. M. Anderson in the Pridine Nucleotide Coenzymes, Academic Press New York, London 1982, J. Everese, B. Anderson, K. You, Editors, Chapter 4. However, most of these derivatives are not accepted well by enzymes. The only derivative which has therefore been previously used for diagnostic tests is 3-acetylpyridine adenine dinucleotide (acetyl NAD) which was first described in 1956 (N. O. Kaplan, J. Biol. Chem. (1956) 221, 823). This coenzyme is also not accepted well by enzymes and exhibits a change in the redox potential.
The use of other NAD derivatives with a modified pyridine group is described in WO 01/94370. However, modifications of the nicotinamide group usually have a direct effect on the catalytic reaction. In most cases this effect is negative.
In another stabilization concept the ribose unit was modified in order to influence the stability of the glycosyl bond. This process does not directly interfere with the catalytic reaction of the nicotinamide group. However, there may be an indirect effect as soon as the enzyme binds strongly and specifically to the ribose unit. In this connection Kaufmann et al. disclose a number of thioribose-NAD derivatives in WO 98/33936 and U.S. Pat. No. 5,801,006 and WO 01/49247. However, a correlation between the modification of the nicotinamide ribose unit and the activity of the derivatives in enzymatic reactions has previously not been demonstrated.
CarbaNAD, a derivative without a glycosyl bond was described for the first time in 1988 (J. T. Slama, Biochemistry 1989, 27, 183 and Biochemistry 1989, 28, 7688). In this derivative the ribose is substituted by a carbacyclic sugar unit. Although carbaNAD was described as a substrate for dehydrogenases, its activity has not yet been proven in clinical biochemical detection methods.
A similar approach was described later by G. M. Blackburn, Chem. Comm., 1996, 2765 in order to synthesize carbaNAD with a methylene bisphosphonate compound instead of the natural pyrophosphate. The methylene bisphosphonate is more stable towards phosphatases and was used as an inhibitor for a ADP ribosylcyclase. The aim was not to make it more resistant to hydrolysis (J. T. Slama, G. M. Blackburn).
Hence the object of the present invention is to provide stable bioanalytical measuring systems especially for determining glucose which avoid the sensitivity to hydrolysis of NAD/NADP and at the same time are active as coenzymes in enzyme reactions.